The clinical application of recombinant adenoviruses as vectors for gene therapy brings about the need to develop new analytical methodologies for monitoring the quality of the viral production as well as establishing structure–function relationships. A mass spectrometry-based assay has been developed for the characterization of structural proteins of the recombinant adenovirus type 5 vector encoding human p53 tumor suppressor gene. The fingerprinting of the viral proteome was accomplished by integration of MALDI-MS and/or MALDI-PSD-MS with SDS–PAGE and RP-HPLC, followed by database search using MS-Fit and MS-Tag algorithms. Viral proteins (molecular weights ∼10,000–100,000Da) corresponding to more than 95% of total protein mass were resolved and identified, which include hexon (II), penton base (III), peripentonal hexon-associated protein (IIIa), minor core protein (V), major core protein (VII), and other hexon-associated proteins (VI and VIII). An important finding of our studies was the identification of some precursor proteins (i.e., pVIII) and propeptides of precursor proteins (pVIII, pX, and pVI) present in the adenovirus sample. The information obtained allows direct and accurate assessment of the quality of recombinant adenoviruses.

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.

A new methodology for the extraction and characterization of proteins from Coomassie-stained sodium dodecylsulfate polyacrylamide gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been described. The utility of this methodology was demonstrated in the characterization of adenovirus proteins. The key steps in the extraction and destaining process involve washing the excised band with a combination of solvents that include 10% acetic acid, acetonitrile, methanol, and formic acid:water:isopropanol mixture. By using this procedure, we determined adenovirus proteins with molecular weights ranging from 10,000 to 110,000 Da by MALDI-MS, obtaining a detection limit of approximately 6 pmol. Parallel experiments were successfully carried out to analyze adenovirus proteins from Cu-stained gels. It was observed that increase in laser intensity resulted in significant improvements in the quality of MALDI mass spectra for the analysis of inefficiently destained proteins from Cu-stained gels.